Effect Of Sperm P34H Expression On Enzyme Activity In Semen And Sperm Fertilization Ability

Objective:To study the correlation between the level of P34H expression and the activity of acrosin,activity of hyaluronidase and activity of neutral a-glucosidase(NAG)in human spermatozoa.To construct an P34H shRNA lentiviral vector and investigate the silencing effects of P34H shRNA on the expression of P34H,acrosome reaction rate,activity of acrosin,activity of hyaluronidase and fertilizing ability of mouse sperm.Methods:1.eighty eight semen samples were collected.Sixty eight cases were in infertile group,and twenty in normal control group.Semen routine analysis was referred to WHO standard method.According to the difference of semen parameters,68 cases of infertile males were divided into infertile group with normal semen parameters and abnormal semen parameters.Real-time PCR was used to detect the level of P34H mRNA expression on spermatozoa.Western blot was used to detect the level of P34H expression on spermatozoa.The P34H-positive rate on human spermatozoa was determined by indirect immunofluorescent staining using anti-P34H antibody.2.The acrosin activity(acrosin-positive rate,acrosin-activity intensity)in all samples were examined by improved fixed-substrate film method.The HYD-positive rate and HYD-activity intensity in all samples were examined by improved fixed-substrate film method.Neutral a-glucosidase activity in semen test kits was used to detect the level of neutral?-glucosidase.3.The recombinant plasmid series of P34H targeted short hairpin RNA(shRNA)were constructed by GV248 plasmids vector.These recombinant plasmids were transformed into DH5a competent cells,and the plasmids were taken from DNA sequencing analysis.The 293T cells were co-transfected with shRNA and lentiviral packaging plasmids.The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to injected into the mouse epididymis and the the expression of P34H at mRNA and protein levels was detected by real-time PCR and Western blotting respectively.The location of P34H protein on mouse spermatozoa was determined by indirect immunofluorescent staining using P34H antibody.4.The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to injected into mice epididymis.The acrosome reaction rate of sperm were detected by CTC staining.The positive rate and activity intensity of acrosin was detected by modified Gelatin membrane.The positive rate and activity intensity of hyaluronidase(HYD)was detected by modified sodium hyaluronate-gelatin membrane.In vitro fertilization was carried out to detect the penetration rate.Results:1.The level of P34H mRNA in infertile groups with normal semen parameters and abnormal semen parameters were lower than that in normal feritile group(p<0.05).The level of P34H protein expression and the percentage of the P34H-positive rate in infertile groups with normal semen parameters and abnormal semen parameters were lower than those in normal feritile group(p<0.05).There were no differences in the level of P34H protein expression and the percentage of the P34H-positive rate between infertile groups with normal semen parameters and abnormal semen parameters(P>0.05).There was no difference in the level of P34H protein expression and the percentage of the P34H-positive rate between normal NAG activity group and abnormal NAG activity group(P>0.05).2.The activity of acrosin(acrosin-positive rate,acrosin-activity intensity)in normal feritile group was significantly higher than that in infertile groups with normal semen parameters and abnormal semen parameters.The activity of HYD(HYD-positive rate,HYD-activity intensity)in infertile groups with normal semen parameters and abnormal semen parameters were also significantly lower than that in control group(P<0.05).The relation between the P34H protein expression and acrosin-positive rate,acrosin-activity intensity had a significant positive correlation(r=0.468,0.310;P<0.01);the relation between the the percentage of P34H-positive rate and acrosin-positive rate,acrosin-activity intensity had a significant positive correlation(r=0.784,0.598;P<0.01).The relation between the P34H protein expression and HYD-positive rate,HYD-activity intensity had a significant positive correlation(r=0.449,0.431;P<0.01);the relation between the the percentage of P34H-positive rate and HYD-positivre rate,HYD-activity intensity had a significant positive correlation(r=0.727,0.691:P<0.01).The relation between the P34H protein expression and neutral a-glucosidase activity had a significant positive correlation(r=0.384,P<0.01);the relation between the the percentage of P34H-positive rate and neutral a-glucosidase activity had a significant positive correlation(r=0.596,P<0.01).3.DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were successfully inserted into the lentiviral vectors.P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels compared with those of the non-transfected and normal control vectors(P<0.05).4.The GV-P34H-shRNA 1 played a significant role in reducing the percentage of P34H positive rate,the positive rate and activity intensity of hyaluronidase in mouse sperm.The silencing effect did not significantly differ between the non-transfected and normal control vectors(P>0.05).The GV-P34H-shRNA 1 played a significant role in reducing acrosome reaction rate,the positive rate of acrosin,activity intensity of acrosin and penetration rate in mouse sperm(P<0.05).The silencing effect did not significantly differ between the non-transfected and normal control vectors(P>0.05).Conclusion:1.The level of P34H protein expression and the percentage of the P34H-positive rate were decreased,while the activity of acrosin(acrosin-positive rate,acrosin-activity intensity),the activity of HYD(HYD-positive rate,HYD-activity intensity)and the activity of neutral a-glucosidase were all reduced in male infertility.2.There was a significant positive correlation between P34H protein expression and acrosin-positive rate,acrosin-activity intensity,HYD-positive rate,HYD-activity intensity,neutral a-glucosidase activity.There was also a significant positive correlations between the percentage of P34H-positive rate and acrosin-positive rate,acrosin-activity intensity,HYD-positive rate,HYD-activity intensity,neutral a-glucosidase activity.5.DNA sequencing analysis confirmed that the three P34H-shRNA sequences were successfully inserted into the lentiviral vectors.P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels.4.P34H-shRNA could significantly inhibit the percentage of P34H positive rate,acrosome reaction rate,the activity of acrosin,the activity of hyaluronidase and penetration rate in mouse sperm.